Visceral leishmaniasis (VL) is characterized by uncontrolled parasitization of spleen, liver, and bone marrow, and is genrally fatal in the absence of treatment. Overall, treatment of VL remain unsatisfactory, and there is an urgent need to develop new chemotherapies, immunotherapies, or combination regimens to improve the efficacy of current drugs and to prevent drug resistance. IL-10 has been implicated in the suppression of potentially curative antigen-specific Th1 cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing killing of Leishmania donovani in murine models of VL. Importantly, the source of the elevated IL-10 in human VL is not known. IL-10 produced by naturally occurring C4+CD25+Foxp3+ regulatory T cells has been shown to promote chronic infection in mouse models of cutaneous leishmaniasis, and accumulating findings pertaining to other chronic infections in humans have implicated natural Treg in the down-modulation of protective immunity. The primary objective of the proposed studies is to evaluate the contribution of naturally occurring Treg, as well as CD4+ and CD8+ adaptive Treg subsets that are induced by antigen encounter in the periphery, to IL-10 production in human VL, and their association with the pathogenesis of this disease. We have initiated and propose to extend a series of clinical studies that are greatly strengthened by 1) large sample size, 2) analysis of cells from peripheral blood as well as splenic aspirates, a primary target organ in VL, 3) comparisons of pre-and post-treatment samples, as well as PBMC from healthy endemic controls, and 4) analysis of both cytokine mRNA expression and protein. Flow cytometry and real-time PCR will be used to determine the frequency and cytokine profiles of Treg subsets, and culture conditions using splenic cells and PBMC will be established to provide direct evidence for the immunosuppressive function of the cells and cytokines, and to screen recombinant vaccine candidate antigens for their capacity to differentially induce effector or regulatory T cell responses. While the immunologic defects associated with human VL are bound to be multi-factorial, a full scale investigation into the role of regulatory T cells and cytokines is warranted based on the accumulating experimental and clinical evidence, and the need to carefully define the subsets involved and their mode of action prior to targetting the cells or cytokines as a therapeutic strategy.